PROJECT ABSTRACT. Somatic activating mutations in the kinase domain of the epidermal growth factor receptor (EGFR) drive the growth of 10-20% of non-small cell lung cancers (NSCLCs), the leading cause of cancer mortality1,2. EGFR tyrosine kinase inhibitors (TKIs) are effective in many EGFR-mutant NSCLC patients1,2. However, this clinical efficacy is limited by innate, adaptive, and acquired EGFR TKI resistance that prevents long-term patient survival3-6. Identifying the mechanisms that limit EGFR TKI response is essential to improve clinical outcomes. When EGFR-mutant patients do respond to initial EGFR TKI treatment, the responses are typically incomplete because some tumor cells persist and survive as residual disease through poorly understood mechanisms5,7-10. These residual disease cells form a reservoir of EGFR TKI-tolerant cells that eventually grow to cause acquired resistance. There is an urgent need to define the molecular events that allow these EGFR-mutant tumor cells to persist as residual disease during initial EGFR TKI treatment in order to design therapeutic strategies to intercept this process and thereby improve the magnitude and duration of EGFR TKI response in patients. From its inception, the goals of this project have been to: (a) understand the mechanism(s) by which NF-kB limits EGFR TKI response and (b) identify a small molecule inhibitor of NF-kB that can effectively and safely enhance EGFR TKI response in EGFR-mutant NSCLC. In ongoing studies funded by this project, we have made progress towards achieving these goals7,11-20. Our initial studies demonstrated NF-kB is a promising target to overcome innate and prevent acquired EGFR TKI resistance, through experiments that revealed a novel role for EGFR TKI-induced adaptive activation of NF-kB in driving incomplete response and the residual disease that is a prelude to acquired resistance. We showed the novel direct NF-kB inhibitor, PBS-1086, can be combined safely with an EGFR TKI to enhance response, suppress residual disease, and prevent acquired resistance in preclinical EGFR-mutant NSCLC cellular and animal models7. Based on our work, PBS-1086 is undergoing clinical development. We now seek to extend this project in novel directions to dissect the mechanism by which NF-kB mediates tolerance to EGFR TKI treatment to promote the residual disease that fuels acquired resistance. We will test the innovative hypothesis that NF-kB drives unexplained and emerging features of drug-tolerant persister cells during EGFR TKI treatment: (1) apoptotic resistance (Aim 1) and (2) de novo gain of EGFR TKI resistance mutations (Aim 2)7-9,21. These persister cell features arising via NF-kB activation could be mutually reinforcing by simultaneously enabling tumor cell plasticity, survival, and genetic adaptation to promote the evolution of EGFR TKI resistance. We are pursuing a long-term strategy to define the function of NF-kB in limiting response to EGFR TKI treatment to guide future efforts to rationally deploy inhibitors of NF-kB signaling or of its key targets in order to better constrain the evolution of resistance and improve clinical outcomes.